Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that leads to severe disability and death. Of the current FDA-approved medications for relapsing forms of MS, all have significant side effects. We propose to use a multi-disciplinary, structure-based approach to developing novel polypeptide blockers of the voltage-gated potassium channel Kv1.3 as new interventions for MS. This channel is highly expressed by terminally-differentiated effector memory T (TEM) lymphocytes that play a major role in MS pathogenesis. Extensive in vitro and in vivo efficacy and safety studies have validated Kv1.3 as a target for immunotherapy and shown that the peptide ShK, originally isolated from a sea anemone, is not only a potent blocker of this channel but also an effective immunomodulator. However, its lack of selectivity for Kv1.3 channels creates a high risk of toxicity through interactions with other potassium channels. We have developed a first generation of synthetic analogs of ShK by modifying its N-terminus with non-protein adducts. These analogs show enhanced specificity for Kv1.3 over other ion channels while retaining picomolar potency, and they selectively suppress cytokine production and proliferation of human TEM cells without affecting other T cell subsets. In rat studies, one of these analogs (ShK-186) suppresses TEM cell enlargement and motility in inflamed tissues, inhibits delayed-type hypersensitivity, and effectively treats chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE; a model of MS) and pristane-induced arthritis. While ShK-186 and related analogs have an excellent safety profile in rats and do not compromise the protective immune response to acute infection with viral (influenza) or bacterial (chlamydia) pathogens, they suffer from several limitations: (i) they are sensitive to changes in pH and temperature; (ii) they have very short in vivo half-lives; (iii) a phosphorylated residue on ShK-186 can be dephosphorylated; (iv) they contain a Met residue that is susceptible to oxidation; and (v) their non-protein adducts are immunogenic. We now propose to design, generate, and evaluate novel analogs of ShK. Under Specific Aim 1 we will use molecular modeling and high-resolution NMR to determine the docking configuration of ShK analogs on Kv1.3 and thereby to design and synthesize more potent and selective N- and C-terminally extended ShK analogs. Under Specific Aim 2 we will assess ShK analogs for their in vitro potency, selectivity, stability, and effects on T lymphocyte activation, and PEGylate them to increase their circulating half-life. Under Specific Aim 3 we will evaluate the most potent and selective ShK analogs in vivo for pharmacokinetics, immunogenicity, safety, and efficacy. This project will generate novel peptide blockers of Kv1.3, which we believe will be valuable leads in the development of new treatments for MS and other chronic inflammatory diseases.